AT7519

Catalog No.S1524 Batch:S152402

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Technical Data

Formula

C16H17Cl2N5O2
Molecular Weight 382.24 CAS No. 844442-38-2
Solubility (25°C)* In vitro DMSO 25 mg/mL (65.4 mM)
Water Insoluble
Ethanol Insoluble
In vivo (Add solvents to the product individually and in order)
Clear solution
2%DMSO 30%PEG300 2%Tween80 66%ddH2O
1.0mg/ml Taking the 1 mL working solution as an example, add 20 μL of 50 mg/ml clarified DMSO stock solution to 300 μL of PEG300, mix evenly to clarify it; add 20 μL of Tween80 to the above system, mix evenly to make it clear; then continue to add 660 μL of ddH2O to adjust the volume to 1 mL. The mixed solution should be used immediately for optimal results. 
* <1 mg/ml means slightly soluble or insoluble.
* Please note that Selleck tests the solubility of all compounds in-house, and the actual solubility may differ slightly from published values. This is normal and is due to slight batch-to-batch variations.
* Room temperature shipping (Stability testing shows this product can be shipped without any cooling measures.)

Preparing Stock Solutions

Biological Activity

Description AT7519 is a multi-CDK inhibitor for CDK1, 2, 4, 6 and 9 with IC50 of 10-210 nM. It is less potent to CDK3 and little active to CDK7. AT7519 also decrease GSK3β phosphorylation. AT7519 induces apoptosis. Phase 2.
Targets
CDK9/CyclinT [1]
(Cell-free assay)		 CDK5/p35 [1]
(Cell-free assay)		 CDK2/CyclinA [1]
(Cell-free assay)		 GSK-3β [1]
(Cell-free assay)		 CDK4/CyclinD1 [1]
(Cell-free assay)		 View More
<10 nM 13 nM 47 nM 89 nM 100 nM
In vitro AT7519 is an ATP competitive CDK inhibitor with a Ki value of 38 nM for CDK1. AT7519 is inactive against all non-CDK kinases with the exception of GSK3β (IC50 = 89 nM). AT7519 shows potent antiproliferative activity in a variety of human tumor cell lines with IC50 values ranging from 40 nM for MCF-7 to 940 nM for SW620 consistent with the inhibition of CDK1 and CDK2. [1] AT7519 induces dose-dependent cytotoxicity in multiple myeloma (MM) cell lines with IC50 values ranging from 0.5 to 2 μM at 48 hours, with the most sensitive cell lines being MM.1S (0.5 μM) and U266 (0.5 μM) and the most resistant MM.1R (>2 μM). It does not induce cytotoxicity in peripheral blood mononuclear cells (PBMNC). AT7519 partially overcomes the proliferative advantage conferred by IL6 and IGF-1 as well as the protective effect of bone marrow stromal cells (BMSCs). AT7519 induces rapid dephosphorylation of RNA pol II CTD at serine 2 and serine 5 sites, and leads to the inhibition of transcription, partially contributing to AT7519 induced cytotoxicity of MM cells. AT7519 induces activation of GSK-3β by down-regulating GSK-3β phosphorylation, which also contributes to AT7519 induced apoptosis independent of the inhibition of transcription. [2]
In vivo A twice daily dosing of AT7519 (9.1 mg/kg) causes tumor regression of both early-stage and advanced-stage s.c. tumors in the HCT116 and HT29 colon cancer xenograft models. [1] AT7519 treatment (15 mg/kg) inhibits tumor growth and prolongs the median overall survival of mice in the human MM xenograft mouse model in association with increased caspase 3 activation. [2]

Protocol (from reference)

Kinase Assay:[1]
  • In vitro Kinase Assays

    Kinase assays for CDK1, CDK2 and GSK3-β are all carried out in a radiometric filter binding format. Assays for CDK5 are in DELFIA format and for CDKs 4 and 6 in ELISA format. For CDKs 1 and 2, the relevant CDK and 0.12 μg/mL Histone H1 are incubated in 20 mM MOPS, pH 7.2, 25 mM β-glycerophosphate, 5 mM EDTA, 15 mM MgCl2, 1 mM sodium orthovanadate, 1 mM DTT, 0.1 mg/mL BSA, 45 μM ATP (0.78 Ci/mmol) and different concentrations of AT7519 for 2 or 4 hours respectively. For GSK3-β, the relevant enzyme and 5 μM glycogen synthase peptide 2 along with 10 mM MOPS pH 7.0, 0.1 mg/mL BSA, 0.001% Brij-35, 0.5% glycerol, 0.2 mM EDTA, 10 mM MgCl2, 0.01% β-mercaptoethanol, 15 μM ATP (2.31 Ci/mmol) and different concentrations of AT7519 are incubated for 3 hours. Assay reactions are stopped by adding an excess of orthophosphoric acid and filtered using Millipore MAPH filter plates. The plates are then washed, scintillant added and radioactivity measured by scintillation counting on a Packard TopCount. For CDK5, CDK5/p35 and 1μM of a biotinylated Histone H1 peptide (Biotin-PKTPKKAKKL) are incubated in 25 mM Tris-HCl, pH 7.5, 2.5 mM MgCl2, 0.025% Brij-35, 0.1 mg/mL BSA, 1 mM DTT, 15 μM ATP and different concentrations of AT7519 for 30 minutes. Assay reactions are stopped using EDTA, transferred to Neutravidin-coated plates and phosphorylated peptide quantified by means of a rabbit phospho-cdk1 substrate polyclonal antibody and DELFIA europium-labelled anti-rabbit IgG secondary antibody using time-resolved fluorescence at λex=335nm, λem=620nm. For CDK 4 and 6 assays, plates are coated with GST- pRb769-921 and blocked with Superblock. CDK4 or 6 is incubated with 15 mM MgCl2, 50 mM HEPES, pH 7.4, 1 mM DTT, 1 mM EGTA, pH 8.0, 0.02% Triton X-100, 2.5% DMSO and different concentrations of AT7519; the reaction is initiated by addition of ATP. After 30 minutes, reactions are stopped by the addition of 0.5 M EDTA pH 8.0. Plates are then washed and incubated for one hour with the primary antibody (anti- p-Rb Serine 780) diluted in Superblock followed by secondary antibody (alkaline phosphatase linked anti-rabbit) for a further hour. Plates are developed using the Attophos system and fluorescence read on a Spectramax Gemini plate reader at excitation 450 nm and emission 580 nm. In all cases, IC50 values are calculated from replicate curves, using GraphPad Prism software.
Cell Assay:[2]
  • Cell lines

    MM.1S, MM.1R, RPMI8226, U266, RPMI8266, RPMI-Dox40, OPM1 cells, primary MM cells and PBMNCs

  • Concentrations

    Dissolved in DMSO at a concentration of 10 mM, final concentrations 0.25-4 μM

  • Incubation Time

    24 or 48 hours

  • Method

    Cells are incubated with different concentrations of AT7519 for 24 or 48 hours at 37 °C. Cell viability is assessed by measuring 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyl tetrasodium bromide (MTT) dye absorbance. DNA synthesis is measured by tritiated thymidine uptake (3H-TdR). Apoptosis is assessed by using Annexin V/PI staining. The percentage of cells undergoing apoptosis is defined as the sum of early apoptosis (Annexin V-positive cells) and late apoptosis (Annexin V-positive and PI-positive cells
Animal Study:[2]
  • Animal Models

    Male SCID mice inoculated subcutaneously with MM.1S cells

  • Dosages

    15 mg/kg/day

  • Administration

    Dosed i.p.

Customer Product Validation

Data from [Data independently produced by AIDS, 2014, 28(15), 2213-22]

Data from [Data independently produced by PLoS One, 2011, 6(9), e25683]

, , Dr. Srinivas Narasipura of Rush University Medical Center

Selleck's AT7519 has been cited by 51 publications

Identification of Selective ATP-Competitive CMG Helicase Inhibitors for Cancer Intervention that Disrupt CMG-Replisome Function [ Res Sq, 2023, rs.3.rs-3182731] PubMed: 37609279
Integrative analysis of drug response and clinical outcome in acute myeloid leukemia [ Cancer Cell, 2022, S1535-6108(22)00312-9] PubMed: 35868306
Non-catalytic allostery in α-TAT1 by a phospho-switch drives dynamic microtubule acetylation [ J Cell Biol, 2022, 221-11e202202100] PubMed: 36222836
Interleukin-15 enhanced the survival of human γδT cells by regulating the expression of Mcl-1 in neuroblastoma [ Cell Death Discov, 2022, 8(1):139] PubMed: 35351861
Modulation of Primary Cilia by Alvocidib Inhibition of CILK1 [ Int J Mol Sci, 2022, 23(15)8121] PubMed: 35897693
O-GlcNAc transferase maintains metabolic homeostasis in response to CDK9 inhibition [ Glycobiology, 2022, cwac038] PubMed: 35708495
Cdk2 suppresses IL-23 expression and the onset of severe acute pancreatitis [ Immun Inflamm Dis, 2022, 10(6):e631] PubMed: 35634959
Biochemical and structural insights into SARS-CoV-2 polyprotein processing by Mpro [ Sci Adv, 2022, 8(49):eadd2191] PubMed: 36490335
KAP1 is a new non-genetic vulnerability of malignant pleural mesothelioma (MPM) [ NAR Cancer, 2022, 4(3):zcac024] PubMed: 35910692
Phosphorylation of a Human Microprotein Promotes Dissociation of Biomolecular Condensates [ J Am Chem Soc, 2021, 10.1021/jacs.1c05386] PubMed: 34346674

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SHIPPING AND STORAGE
Selleck products are transported at room temperature. If you receive the product at room temperature, please rest assured, the Selleck Quality Inspection Department has conducted experiments to verify that the normal temperature placement of one month will not affect the biological activity of powder products. After collecting, please store the product according to the requirements described in the datasheet. Most Selleck products are stable under the recommended conditions.

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